Molecular Epidemiological Research Of Chonorchis Sinensis Infection In Fuyu City,Jilin Province

Clonorchiasis is a serious parasitic zoonosis which is also called liver fluke disease. It is a kind of foodborne disease caused by Clonorchis sinensis parasitizing in bile duct of human and other mammals. People usually get infected with Clonorchis sinensis by eating raw or undercooked fish with its metacercaria inside. And then metacercaria will develop into adult and spawn in the infected people. East Asia and Southeast Asia are the major regions of clonorchiasis. There are about more than 30 millions infected people around the world, and about half of them are Chinese. So China is one of the most serious country against Clonorchis sinensis infection. Jilin Province is a high-risk area of clonorchiasis in China, and the Fuyu city in Jilin is one of the epidemiological areas along the Songhua River basin. So it is imperative to undertake this epidemiological investigation in Fuyu City. Meanwhile the molecular biological analysis of isolates in this area needs to be done to get the clear isolate of species. It is significant to understand the species specificity. Therefore, we did this molecular epidemiological research of Clonorchis sinensis infection in Fuyu City.(1) Epidemiological investigation in Fuyu city along river basinWe had 2142 villagers from 14 villages of 3 towns in Fuyu City completed the questionnaire survey, and collected 1745 pieces of stool samples. After screening the stool samples by Kato-Katz thick smear method, we got 513 pieces of positive stool samples and 513 serums. After the final lab screening, these serums will provide the positive serum specimens for the research of laboratory diagnostic kit. Statistical method was used to analyse the relevant data(gender, age, food habits about raw fish, drink) from questionnaires. The result showed the rate of infection was 36.2% for male and 21.3% for female, with significant difference(P<0.01); age of 40 to 59 was the major group of infected people; the food habits about raw fish(OR=3.211) and drink(OR=1.97) also showed significant impact(P<0.01). We also had the 513 villagers to take ultrasonic examination to further understand the imaging diversity after infected with Clonorchis sinensis, such as thickening of intrahepatic bile duct wall, which were very possible caused by infection. The result showed diffuse dilatation of the small intrahepatic bile ducts and thickening of the intrahepatic bile duct were observed in 18% of infected people. Sonogram of gallbladder disclosed several discrete, fusiform, floating and dependent echogenic foci in 2.5% of infected people. In the meantime, we investigated the local environment and did metacercaria detection against 10 kinds of freshwater fishes and shrimp from the fresh markets of 3 towns in Fuyu. The result showed that Pseudorasbora parva,Whitefish,Saurogobio dabryi had the higher rate of metacercaria infection than other kinds of fishes. We got conclusions by this epidemiological investigation: 1).Kato-Katz thick smear method is still a quick and major way for large-scale investigation of Clonorchis sinensis. 2). Eating raw fish/shrimp is an important way to get infected with Clonorchis sinensis; Male, elder and drinking alcohol get higher infection rate. 3). Ultrasonic examination can be used as a simple auxiliary examination for diagnosis of clonorchiasis. 4). The basic measures to control this disease include improving environmental sanitation and correcting eating habits.(2) The design and screening of specific primers of Clonorchis sinensisDuring the metacercaria detection against the fishes from local fresh markets, two kinds of metacercaria like as Clonorchis sinensis and Metorchis orentalis were observed. To confirm the species based on the genetic level, molecular biology research was done. To confirm all eggs we collected during this epidemiological investigation belonging to Clonorchis sinensis, we looked for the conservative regions by comparing with the sequences in Genbank, designed pairs of primers and done the screening. We found out 2 pairs of primers Cs F2R3 and Mo F2R2 as the specific primers for Clonorchis sinensis and Metorchis orentalis respectively by using common PCR and multiple PCR. After sequencing, the amplicons had high homology with clonorchis sinensis and metorchis orentalis respectively.(3) The establishment and optimization of molecular biological diagnosis assay for ClonorchiasisUsed the specific primers to establish the reaction system, and optimized the reaction condition including primers, template concentration and reaction time etc. And at last,we got the optimalizing reaction system and reaction condition. The purpose is to increase the sensitivity of diagnosis, and provide technical support to the gene amplification of eggs from stool samples. Through the repeated research, we finally got the molecular biological diagnosis assay by using the DNA of Clonorchis sinensis as template successfully.(4) Isolate identification and nucleic acid sequence analysis of the Clonorchis sinensis along Songhua River basin in Fuyu CityTo understand the genotype of Clonorchis sinensis along Songhua River basin, we identified species and genotypes by mutiple PCR of the DNA of eggs from stool samples with the specific primers of Clonorchis sinensis and Metorchis orentalis we screened out. The result showed there were no Metorchis orentalis infection in the 513 positive eggs stool samples. And 265 samples of them(51.7%) showed the eggs of Clonorchis sinensis. By comparing the sequence by BLAST, the result revealed that Clonorchis sinensis ITS2 had 99%-100% of homology with AF217099 and AF217094. Conclusion: PCR method is a useful and definite tool to increase the accuracy of diagnosis of clonorchiasis, and decrease the misdiagnosis rate; we collected the standard positive serum of patients of Clonorchiasis, which would provide an important specimen support to the development of diagnostic kit.